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Shermado, Fairouz M. (2014) Molecular Characterization of Cannabinoids and Free Fatty Acid Receptors in Human and Rat Skeletal Muscle

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Shermado, Fairouz M. (2014) Molecular Characterization of Cannabinoids and Free Fatty Acid Receptors in Human and Rat Skeletal Muscle Shermado, Fairouz M. (2014) Molecular characterization of cannabinoids and free fatty acid receptors in human and rat skeletal muscle. PhD thesis, University of Nottingham. Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/14145/1/Fairouz_Shermado.pdf Copyright and reuse: The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. · Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. · To the extent reasonable and practicable the material made available in Nottingham ePrints has been checked for eligibility before being made available. · Copies of full items can be used for personal research or study, educational, or not- for-profit purposes without prior permission or charge provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. · Quotations or similar reproductions must be sufficiently acknowledged. Please see our full end user licence at: http://eprints.nottingham.ac.uk/end_user_agreement.pdf A note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. For more information, please contact [email protected] MOLECULAR CHARACTERISATION OF CANNABINOID AND FREE FATTY ACID RECEPTORS IN HUMAN AND RAT SKELETAL MUSCLE Fairouz M. Shermaddo, MBChB, MSc Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy September 2013 Abstract The mechanisms underlying the development of insulin resistance in skeletal muscle are very complex and are not completely understood. Insulin resistance in skeletal muscle is of particular importance because muscle is the major site of insulin stimulated glucose uptake. Skeletal muscle is one of the major insulin sensitive organs and it is responsible for 80% of insulin stimulated glucose disposal as well as fatty acid oxidation. Elevated circulating free fatty acids (FFAs) and their derivatives such as endocannabinoids (ECs) have been described in obesity and are thought to be influential in the development of muscle insulin resistance. While several hypotheses have been put forward to explain the mechanisms by which FFAs and ECs may cause insulin resistance, there are still many potential signalling pathways which may be involved that have not yet been examined. The main aim of this thesis was to characterize the role of the cannabinoid receptors and free fatty acid receptor 1 (GPR40) in cell signalling in human and rat skeletal muscle tissue and primary cultured myotubes. Gene expression profiling of human skeletal muscle and cultured myotubes and myoblasts indicated that the cannabinoid receptor CB1 and GPR40 were expressed at low levels and these results were confirmed using Taqman QRTPCR. CB2 receptor expression was only detected in rat tissue and as a result was not further studied in cell culture systems. When global gene expression profiles were further examined it was evident that whilst cultured myotubes retained many characteristics of skeletal muscle tissue, the phenotype appeared to be closer to fetal than adult muscle. Furthermore, when metabolic gene expression networks were analysed using pathway based analysis, it was apparent that expression of genes involved in oxidative phosphorylation, insulin signalling and glucose transport were markedly reduced in cultured cells. The most striking example being GLUT4 which was expressed at approximately 3000-fold lower levels in cell culture as compared to tissue. i The functionality of CB1 and GPR40 receptors was demonstrated using selective agonists and antagonists. CB1 activation by both synthetic and endogenous ligands was confirmed using phosphorylation of ERK1 and 2 as was the presence of functional GPR40 protein in myotubes. Neither agonists nor antagonists of CB1 or GPR40 receptors were found to modulate insulin signalling as determined by phosphorylation of downstream targets Akt and GSK3/ Global expression profiling was also carried out on myotubes treated with GPR40 agonists and antagonists obtained from AstraZeneca. No changes in metabolic or insulin signalling genes were observed. Rather, antagonists of GPR40 appear to activate gene expression networks involved in cell proliferation – in particular an elevation in the ERBB2 signalling pathway. ii Poster presentation Fairouz Shermado, Kostas Tsintzas and Andrew J. Bennett. Cannabinoid Receptors in Human and Rat (Wistar and Zucker) Skeletal Muscle. 6th European Workshop on Cannabinoid Research, 2013, Dublin, Ireland. Fairouz Shermado, Kostas Tsintzas and Andrew J. Bennett. Characterization and Functional Significance of Cannabinoid Receptors in Skeletal Muscle. BPS, Winter meeting, 2012, London, UK iii Acknowledgements Praise be to Allah I would like to thank my primary supervisor Dr Andrew Bennett for the expert guidance, and support throughout my PhD studies. I am also grateful to Dr Kostas Tsintzas for the additional supervisory support and encouragment. Thanks also to Dr. Steve Alexander for readily offering help and advice whenever called upon. I am eternally grateful for the help and support from friends and colleagues in the FRAME lab. Special thanks to Monika Owen and Roya Jaddi for the technical and social support. I recognize that this research would not have been possible without the financial assistance of Libyan Higher Education. To my mother, sisters and brothers I say, thanks for believing in me. Your prayers, words of encouragement and support have been very helpful in getting me here. You kept me going when the going seemed tough and insurmountable. I continue to realize and appreciate in new ways how fortunate I am to have such family. To my husband, thanks for your immense help, love and solid support, without your encouragement and immeasurable assistance I would not have finished this thesis. I wish you the best for your PhD study. To my adorable daughters (Alla and Asma) and sons (Mohamed and Ahmed) I say, sorry for not spending much time with you, breaking my promises (occasionally!) and thanks for your love. I now hope we can enjoy our family life and I will do my best to be “a real mum who cares about her children more than her work” as you wished once. I would like to dedicate this thesis to the soul of my father who had always been, and continue to be, my inspiration. I could never adequately express all that he has given to me, or all that he has meant to me. Above all, glory and honour go to God almighty, for granting me the strength, the will to succeed even in the face of adversity, and for his abundant mercies and blessings upon my life. iv Declaration I hereby declare that the work presented in this thesis, has not been submitted for any degree or diploma, at this, or any other university, and that all of the experiments, unless otherwise stated, were performed by me. Dr. Fairouz M. Shermado v Contents Abstract ............................................................................................................... i Poster presentation ............................................................................................ iii Acknowledgements ............................................................................................ iv Declaration .......................................................................................................... v Contents ............................................................................................................. vi List of Figures ..................................................................................................... x List of Tables ...................................................................................................... xv List of Abbreviations ......................................................................................... xvi IKK: IB kinase ........................................................................................... xvii CHAPTER 1. General Introduction ................................................................... 1 1.1. Insulin resistance in skeletal muscle ..................................................... 1 1.1.1. Introduction ............................................................................................. 1 1.1.2. Mechanisms underlying the insulin resistance induced by saturated fatty acids . 2 1.2. The endocannabinoid system............................................................... 11 1.2.1. Introduction ............................................................................................ 11 1.2.2. Endocannabinoids synthesis and degradation .............................................. 11 1.2.3. Mechanism of action of endocannabinoids ................................................... 13 1.2.4. Other Potential endocannabinoid receptors: ................................................ 14 1.2.5. The roles of Endocannabinoids .................................................................. 16 1.2.6. Role of endocannabinoids in obesity and type 2 diabetes mellitus (T2D). ....... 21 1.3. FREE FATTY ACID RECEPTORS ............................................................
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